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Ependymoma is a metastatic CNS tumor responsible for 10% of all pediatric brain tumors. Displaying a poor survival rate of only 32.5%, the conventional ependymoma treatment is surgery and radiation therapy associated to significant morbidity. Previous studies found significantly increased expression of NESTIN, a neural progenitor cell (NPC) marker, in aggressive ependymoma. Zika virus (ZIKV) has recently emerged as an oncolytic therapy against adult and pediatric CNS tumors due to its ability to infect NPCs and neural stem-like tumor cells. We showed for the first time that Brazilian Zika virus strain efficiently destroyed pediatric CNS tumor cells in vitro and in vivo, resulting in significantly mice longer survival and fewer metastasis. Once tumor cells overexpressing NPCs markers are more susceptible to ZIKV infection, here we evaluated the oncolytic properties of Brazilian ZIKV against human ependymoma. A patient-derived ependymoma cell line (USP21-EPE) has been established and found to abnormally express the pluripotency markers CD133, SOX2 and NESTIN. USP21-EPE monolayer culture showed massive cell death three days after ZIKV infection in four tested MOIs: 0.01, 0.1, 1 and 2. At MOI 0,1 50% of cell population decreased viability while at higher MOIs was observed 100% of cell death 72 hours post ZIKV infection. Moreover, ZIKV significantly disrupted CNS ependymoma tumorspheres reinforcing its oncolytic effect against the ependymoma tumor cells. Considering the poor effectiveness and severe side effects of available treatments for ependymoma and that most ZIKV infections are associated to very mild or no symptoms, our findings open new avenues for novel therapies against this aggressive pediatric tumor. Citation Format: Carolini Kaid, Patrícia Semedo-Kuriki, Ernesto Goulart, Luiz Caires-Júnior, Renato Astray, Oswaldo Keith Okamoto, Mayana Zatz. Zika virus is a potent oncolytic agent against aggressive human ependymoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2869.

Genetic characterization of protein-producing clones represents additional value to cell line development. In the present study, ten Per.C6 clones producing a Rebmab100 monoclonal antibody were selected using two cloning methods: six clones originated from limiting dilution cloning and four by the automated colony picker ClonePix FL. A stability program was performed for 50 generations, including 4 batches distributed along the timeframe to determine specific productivity (Qp) maintenance. Four stable clones (two from limiting dilution and two from ClonePix FL) were further evaluated. The relative mRNA expression levels of both heavy chain (HC) and light chain (LC) genes were verified at generations 0, 30-35, and 50-55 of the stability program. At generations 0 and 30-35, LC gene expression level was higher than HC gene, whereas at generation 50-55, the opposite prevailed. A high correlation was observed between Qp and HC or LC mRNA expression level for all clones at each generation analyzed along the continuous culture. The mRNA stability study was performed at steady-state culture. The LC gene displayed a higher half-life and lower decay constant than HC gene, accounting for the higher observed expression level of LC mRNA in comparison to HC mRNA. Clone R6 was highlighted due its high Qp, mRNA expression levels, and mRNA stability. Besides the benefits of applying genetic characterization for the selection of stable and high-producing clones, the present study shows for the first time the correlation between Qp and HC or LC expression levels and also mRNA stability in clones derived from human cell line Per.C6(®).

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:26:26Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:26:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-04-01 The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC. Department of Oral Pathology School of Dentistry University of São Paulo, Av. Prof. Lineu Prestes 2227, Cidade Universitária, 05508-000 São Paulo Department of Head and Neck Surgery School of Medicine University of São Paulo, Av. Dr. Arnaldo 455, Cerqueira César, 01246903 São Paulo Head and Neck Surgery Division Heliópolis Hospital Complex, Rua Cônego Xavier 276, Sacomã, 04231-030 São Paulo Department of Head and Neck Surgery Arnaldo Vieira de Carvalho Cancer Institute, Rua Dr Cesário Motta Junior 112, Vila Buarque, 01221020 São Paulo Department of Molecular Biology São José do Rio Preto School of Medicine, Av. Brigadeiro Faria Lima 5416-Vila São Pedro, 15090-000 São José do Rio Preto Department of Genetics and Evolutionary Biology Institute of Biosciences University of São Paulo, R. do Matão, travessa 14 321, Cidade Universitária, 05508-090 São Paulo Department of Genetics Biosciences Institute University of São Paulo, Rua do Matão, trav. 14 321, Cidade Universitária, 05508-090 São Paulo School of Medicine USP, São Paulo Albert Einstein Jewish Teaching and Research Institute (IIEP), São Paulo School of Medicine of São José do Rio Preto São José Dos Campos Dental School UNESP Cancer Institute Arnaldo Vieira de Carvalho, São Paulo School of Dentistry USP, São Paulo Heliópolis Hospital, São Paulo School of Medicine of Ribeirão Preto USP School of Public Health USP, São Paulo University of Vale do Paraíba, São José dos Campos School of Medicine UNIFESP, São Paulo Faculty of Pharmaceutical Sciences of Ribeirão Preto USP, SP São José Dos Campos Dental School UNESP

Pre-clinical studies have supported the use of mesenchymal stem cells (MSC) to treat highly prevalent neurodegenerative diseases such as Parkinson’s disease (PD) but preliminary trials have reported controversial results. In a rat model of PD induced by MPTP neurotoxin, we first observed a significant bilateral preservation of dopaminergic neurons in the substantia nigra and prevention of motor deficits typically observed in PD such as hypokinesia, catalepsy, and bradykinesia, following intracerebral administration of human umbilical cord-derived MSC (UC-MSC) early after MPTP injury. However, surprisingly, administration of fibroblasts, mesenchymal cells without stem cell properties, as a xenotransplantation control was highly detrimental, causing significant neurodegeneration and motor dysfunction independently of MPTP. This observation prompted us to further investigate the consequences of transplanting a MSC preparation contaminated with fibroblasts, a plausible circumstance in cell therapy since both cell types display similar immunophenotype and can be manipulated in vitro under the same conditions. Here we show for the first time, using the same experimental model and protocol, that transplantation of UC-MSC induced potent neuroprotection in the brain resulting in clinical benefit. However, co-transplantation of UC-MSC with fibroblasts reverted therapeutic efficacy and caused opposite damaging effects, significantly exacerbating neurodegeneration and motor deficits in MPTP-exposed rats. Besides providing a rationale for testing UC-MSC transplantation in early phases of PD aiming at delaying disease progression, our pre-clinical study suggests that fibroblasts may be common cell contaminants affecting purity of MSC preparations and clinical outcome in stem cell therapy protocols, which might also explain discrepant clinical results. Electronic supplementary material The online version of this article (doi:10.1007/s12015-011-9256-4) contains supplementary material, which is available to authorized users.

Bone deposition and bone resorption are ongoing dynamic processes, constituting bone remodeling. Some bone tumors, such as osteosarcoma (OS), stimulate focal bone deposition. OS is the most common primary bone tumor in children and young adults. A complex network of genes regulates bone remodeling and alterations in its expression levels can influence the genesis and progression of bone diseases, including OS. We hypothesized that the expression profiles of bone remodeling regulator genes would be correlated with OS biology and clinical features. We used real-time PCR to evaluate the mRNA levels of the tartrate-resistant acid phosphatase (ACP5), colony stimulating factor-1 (CSF1R), bone morphogenetic protein 7 (BMP7), collagen, type XI, alpha 2 (COL11A2), and protein tyrosine phosphatases zeta 1 (PTPRZ1) genes, in 30 OS tumor samples and correlated with clinical and histological data. All genes analyzed, except CSF1R, were differentially expressed when compared with normal bone expression profiles. In our results, OS patients with high levels of COL11A2 mRNA showed worse overall (p = 0.041) and event free survival (p = 0.037). Also, a trend for better overall survival was observed in patients with samples showing higher expression of BMP7 (p = 0.067). COL11A2 overexpression and BMP7 underexpression could collaborate to OS tumor growth, through its central role in bone remodeling process. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1142–1148, 2010

Drug resistance is one of the main limiting steps for the successful treatment of most cancers. Additionally, the presence of cancer cells with stem cell traits favors the process of treatment resistance, tumor recurrence, and spreading. Glutathione (GSH) is a major antioxidant intracellular molecule and has been positively correlated with the development of drug resistance. The purpose of this study was to verify whether GSH depletion potentiates the effect of chemotherapy in stem-like cells from the medulloblastoma cell line Daoy. Cells expressing normal (control tumor cells) or high levels of the pluripotency factor OCT4A (stem-like cells) were treated with a combination of BSO (glutathione inhibitor), cisplatin, and temozolomide (TMZ). Cell viability was assessed through MTT and apoptosis assays. Initially, the concentration of BSO required to deplete the GSH in both Daoy and Daoy-OCT4A cells was established (50 and 58 μM, respectively), as well as the IC50 of each chemotherapeutic drug. Both cells were sensitive to cisplatin (IC50 = 0.494 μM for Daoy and 0.215 μM for Daoy-OCT4A), but resistant to TMZ. The highest dose of TMZ used (500 μM) reduced the viability of Daoy cells to 56%, but only to 80% in Daoy-OCT4A cells, denoting the chemoresistant phenotype of these cancer stem-like cells. Next, the ideal combination of the three compounds was defined. For Daoy cells, it was BSO 50 μM + cisplatin 0.8 μM + TMZ 0.8 μM. As for Daoy-OCT4A cells, it was BSO 58 μM + cisplatin 0.215 μM + TMZ 500 μM. Daoy-OCT4A cells required a higher dose of TMZ than control Daoy cells to attain similar death rates; however, the combined treatment was effective for both cells. When combining these three drugs, a synergistic effect was observed for Daoy-OCT4A cells, reducing cell viability to ~17%, in comparison with treatment of cisplatin+TMZ alone. As for Daoy cells, an additive effect was observed and viability was reduced to ~15%, in comparison with cisplatin+TMZ alone. This indicates that the use of a glutathione inhibitor in combination with cisplatin and TMZ may be useful in the treatment of medulloblastoma, even in the case of aggressive tumors. Similarly, this approach may also be useful in the development of therapeutic strategies for other chemoresistant tumors. Citation Format: Beatriz Dias Barbieri, Marina Marçola, Clarissa Rocha, Oswaldo Keith Okamoto. Glutathione depletion overcomes chemotherapy resistance in aggressive medulloblastoma stem-like cells [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A57.

Biologically distinct populations of neoplastic stem cells have been identified in a variety of human cancers, in which they are associated with the initial steps of tumorigenesis. The intrinsic properties of self-renewal, clonogenicity and multipotency, along with a longer half-life within the body, may render normal adult stem cells more prone to accumulate genetic mutations leading to neoplastic transformation, as predicted by the cancer stem cell hypothesis. Tumor formation is also associated with the pluripotency of embryonic stem cells and may be induced as a consequence of complete dedifferentiation of mature cells, as recently reported for induced pluripotent stem cells. The tumor-initiating cell phenotype may result from genetic alterations affecting the expression of critical genes regulating typical stem cell processes such as self-renewal and pluripotency, in addition to genes determining stem cell senescence or longevity. Detailed genome-wide analysis of cancer stem cells and respective normal counterparts will help elucidate the cellular and molecular nature of tumors, providing fundamental information about the initial steps toward malignant transformation. Devising ways of detecting such genetic and epigenetic alterations and cell populations displaying them would allow medical interventions at the early phases of cancer development, thereby improving the chances of favorable clinical outcomes.

This review discusses some of the impacts that biotechnology, genomics and nanotechnology convergence should have on future cancer management, in particular, the development of innovative diagnostic and therapeutic approaches based on monoclonal antibodies (mAbs) and cancer stem cells. Emergent therapeutic strategies in cancer have been focusing on the use of mAbs to stimulate an immune response against tumors, to block signaling pathways, or to refine delivery of cytotoxic agents. Now that cancer stem cells are being identified and characterized in different tumor types, their relevance to cancer physiopathology is becoming evident, making them natural targets for mAb development. Cancer stem cells are postulated to be responsible for tumor development, metastasis and relapse after conventional therapies. Therefore, mAbs targeting specific antigens and related pathways altered in cancer stem cells should facilitate earlier diagnosis through molecular imaging techniques and more efficient destruction of tumor initiating cells, thus improving clinical outcome.

The prognosis of glioblastomas is still extremely poor and the discovery of novel molecular therapeutic targets can be important to optimize treatment strategies. Gene expression analyses comparing normal and neoplastic tissues have been used to identify genes associated with tumorigenesis and potential therapeutic targets. We have used this approach to identify differentially expressed genes between primary glioblastomas and non-neoplastic brain tissues. We selected 20 overexpressed genes related to cell cycle, cellular movement and growth, proliferation and cell-to-cell signaling and analyzed their expression levels by real time quantitative PCR in cDNA obtained from microdissected fresh tumor tissue from 20 patients with primary glioblastomas and from 10 samples of non-neoplastic white matter tissue. The gene expression levels were significantly higher in glioblastomas than in non-neoplastic white matter in 18 out of 20 genes analyzed: P < 0.00001 for CDKN2C, CKS2, EEF1A1, EMP3, PDPN, BNIP2, CA12, CD34, CDC42EP4, PPIE, SNAI2, GDF15 and MMP23b; and NFIA (P: 0.0001), GPS1 (P: 0.0003), LAMA1 (P: 0.002), STIM1 (P: 0.006), and TASP1 (P: 0.01). Five of these genes are located in contiguous loci at 1p31–36 and 2 at 17q24–25 and 8 of them encode surface membrane proteins. PDPN and CD34 protein expression were evaluated by immunohistochemistry and they showed concordance with the PCR results. The present results indicate the presence of 18 overexpressed genes in human primary glioblastomas that may play a significant role in the pathogenesis of these tumors and that deserve further functional investigation as attractive candidates for new therapeutic targets.

Comparative analysis of cancer stem cells with their neoplastic and non-neoplastic counterparts should help better understand the underlying molecular events leading to transformation and tumor dissemination. Here, we report a molecular signature comprised by genes with exclusive aberrant expression in CD133(+) cells, a reported subpopulation of tumorigenic stem-like cells, isolated from human glioblastomas. Microarrays covering 55,000 transcripts were used to compare gene expression profiles in purified subpopulations of CD133(+) and CD133(-) GBM cells. Sixteen genes, many of which not previously associated with astrocytomas, were found aberrantly expressed in CD133(+) cells, but not in CD133(-), when compared with corresponding non-neoplastic controls. Up-regulation of two of such genes, E2F2 and HOXC9, was detected in a set of 54 astrocytomas of different grades and significantly associated with malignancy. Due to their distinctive expression in CD133(+) cells, the use of E2F2 and HOXC9 as therapeutic targets for tumor eradication is suggested.

Since the seminal studies by Fialkow et al. in the seventies [1] and by Bonnet and Dick in the nineties [2], where an organized tumor cell hierarchy originating from transformed hematopoietic stem cells was found in myeloid leukemias, the cancer stem cell (CSC) model of tumor development drew the attention of the scientific community and became the focus of extensive discussion. This theme gained momentum over the past decade, with the identification of stemlike cells as drivers of tumor initiation, recurrence, and metastasis spread in a variety of nonhematopoietic human cancers. These characteristics postulated them as novel and promising therapeutic targets for cancer, especially in the context of drug-resistant tumors [3]. The origin of these stemlike cells is being extensively studied. In some types of tumors, such as in blood, brain, colon, and skin cancer, it has been shown that adult stem cells are prone to transformation due to a longer half-life within tissues compared to their cell progenies, facilitating the accumulation of oncogenic mutations as a result of prolonged exposure to genotoxic stresses [4]. In other cases, de novo acquisition of stem cell properties may occur in either normal or neoplastic cells due to genetic/epigenetic aberrations [5]. Finally, the CSC model has been put forward to explain why tumors like breast cancer might return years after surgery. In this situation, lower proliferation rate might allow cells to escape from aggressive therapy and allow for genetic/epigenetic evolution. Advances in the field, however, point to varied and more complex models of tumor development than previously envisioned, in which the stemlike phenotype may also be dynamically acquired by cancer cells through interaction with stromal cells and soluble factors [6]. A relevant question timely addressed in this special issue is the understanding of the contribution of resident stem cells to the tumor microenvironment (TME). This is of major relevance since they may significantly influence tumor progression, aggressiveness, and response to therapy. The contribution of pericytes to tumor growth, angiogenesis, metastasis, and evasion of immune destruction, which are classic hallmarks of cancer, is comprehensively reviewed by A. L. Ribeiro and O. K. Okamoto. In their article, the authors also discuss a pericyte-mediated regulation of stemness properties in cancer cells and argue in favor of pericytes as cellular targets for new cancer therapies aiming at the TME. Indeed, many new anticancer therapies targeting the TME are under development, with currently approved antiangiogenic drugs as examples of such strategy. However, events of tumor recurrence and poor response to antiangiogenic therapy still puzzle researchers and emphasize the need for continuous studies. In the article by M. Marcola and C. E. Rodrigues, the involvement of endothelial progenitor cells in tumor angiogenesis is critically reviewed. In their article, the specific roles of different members of the VEGF family of growth factors, as well as the relevance of the vascular niche to stemness in cancer cells, are also carefully discussed. Another practical issue of clinical relevance is the rigorous evaluation of potential oncogenic risks associated with stem cell therapy. Long-term safety issues must be properly addressed before stem cell-based therapies enter clinical trials, but such studies are outnumbered in the literature by studies evaluating therapeutic effects based on restoration of tissue integrity and physiological balance. While the original article by T. Jazedje et al. addresses this issue at the preclinical stage, showing in a murine breast adenocarcinoma model that mesenchymal stromal cells (MSC) may exert either pro- or antitumorigenic effects depending on the experimental condition, R. Schweizer et al. bring a more clinical perspective to this issue, presenting an interesting discussion about the possible oncogenic hazards of adipose-derived MSC in breast cancer patients subjected to breast reconstruction after mastectomy. Finally, due to their tumor homing properties, the use of stem cells as vehicles to deliver suicide genes is a strategy highly pursued in cancer gene therapy. However, low delivery efficiency and off-target effects are common limitations of current expression systems. In the original article by Y. Luo et al., the authors elegantly characterize a novel inducible transgene expression system based on the action of neural stem cells that could be further explored to treat malignant gliomas. In summary, the articles compiled in this special issue highlight how the growing investigation of tumor development through the lens of stem cell biologists is significantly impacting basic and translational cancer research. This interplay between stem cell and tumor biology offers an exceptional opportunity to improve our knowledge about cancer, one of the leading causes of death worldwide. Advances in this field are expected to bring significant impacts in cancer diagnosis and therapy. Oswaldo Keith Okamoto Ander Matheu Luca Magnani

Pericytes are multipotent perivascular cells whose involvement in vasculature development is well established. Evidences in the literature also suggest that pericytes display immune properties and that these cells may serve as anin vivoreservoir of stem cells, contributing to the regeneration of diverse tissues. Pericytes are also capable of tumor homing and are important cellular components of the tumor microenvironment (TME). In this review, we highlight the contribution of pericytes to some classical hallmarks of cancer, namely, tumor angiogenesis, growth, metastasis, and evasion of immune destruction, and discuss how collectively these hallmarks could be tackled by therapies targeting pericytes, providing a rationale for cancer drugs aiming at the TME.

The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual ovarian cancer with 211At-Rebmab200. Here, the biodistribution of 211At-Rebmab200 was evaluated, as was the utility of 99mTc-Rebmab200 for bioimaging. Rebmab200 was directly compared with its murine counterpart MX35 in terms of its in-vitro capacity for binding the immobilized NaPi2B epitope and live cells; we also assessed its biodistribution in nude mice carrying subcutaneous OVCAR-3 tumors. Tumor antigen and cell binding were similar between Rebmab200 and murine MX35, as was biodistribution, including normal tissue uptake and in-vivo tumor binding. We also demonstrated that 99mTc-Rebmab200 can be used for single-photon emission computed tomography of subcutaneous ovarian carcinomas in tumor-bearing mice. Taken together, our data support the further development of Rebmab200 for radioimmunotherapy and diagnostics.

In medulloblastoma, abnormal expression of pluripotency factors such as LIN28 and OCT4 has been correlated with poor patient survival. The miR-302/367 cluster has also been shown to control self-renewal and pluripotency in human embryonic stem cells and induced pluripotent stem cells, but there is limited, mostly correlational, information about these pluripotency-related miRNA in cancer. We evaluated whether aberrant expression of such miRNA could affect tumor cell behavior and stem-like traits, thereby contributing to the aggressiveness of medulloblastoma cells. Basal expression of primary and mature forms of miR-367 were detected in four human medulloblastoma cell lines and expression of the latter was found to be upregulated upon enforced expression of OCT4A. Transient overexpression of miR-367 significantly enhanced tumor features typically correlated with poor prognosis; namely, cell proliferation, 3-D tumor spheroid cell invasion and the ability to generate neurosphere-like structures enriched in CD133 expressing cells. A concurrent downregulation of the miR-367 cancer-related targets RYR3, ITGAV and RAB23, was also detected in miR-367-overexpressing cells. Overall, these findings support the pro-oncogenic activity of miR-367 in medulloblastoma and reveal a possible mechanism contributing to tumor aggressiveness, which could be further explored to improve patient stratification and treatment of this important type of pediatric brain cancer.

Misexpression of stem cell-related genes may occur in some cancer cells, influencing patient's prognosis. This is the case of medulloblastoma, a common and clinically challenging malignant tumor of the central nervous system, where expression of the pluripotency factor, OCT4, is correlated with poor survival. A downstream target of OCT4, L1TD1 (LINE-1 type transposase domain-containing protein 1 family member), encodes a novel embryonic stem cell (ESC)-related protein involved in pluripotency and self-renewal of ESCs. L1TD1 is still poorly characterized and its expression pattern and function in cancer cells are virtually unknown. Although normally restricted to non-neoplastic undifferentiated cells and germ cells, we found that high L1TD1 expression also occurs in medulloblastoma cells, reaching levels similar to those found in ESCs, and is correlated with poor prognosis. Conversely to what is reported during normal cell differentiation, when differentiated cells remain healthy, despite L1TD1 downregulation, depletion of L1TD1 protein levels by targeted gene silencing significantly reduced medulloblastoma cell viability, inhibiting cell proliferation and inducing apoptosis. More strikingly, L1TD1 depletion downregulated expression of the neural stem cell markers, CD133 and Nestin, inhibited neurosphere generation capability, and sensitized medulloblastoma cells to temozolomide and cisplatin, two chemotherapeutic agents of clinical relevance in medulloblastoma treatment. Our findings provide insights about the contribution of pluripotency-related genes to a more aggressive tumor phenotype through their involvement in the acquisition of stem-like properties by cancer cells and point out L1TD1 as a potential therapeutic target in malignant brain tumors.

Microarray studies have revealed the differential expression of several genes in mantle cell lymphoma (MCL), but it is unknown which of these differences are dependent on the transformed MCL cell itself or on the tumour microenvironment. To investigate which genes and signalling pathways are aberrantly expressed in MCL cells we used oligonucleotide microarrays to perform gene expression profiling of both purified leukaemic MCL cells and their normal counterparts, the naive B cells. A total of 106 genes were differentially expressed at least threefold in MCL cells compared with naive B cells; 63 upregulated and 43 downregulated. To validate the microarray results in a larger set of samples, we selected 10 differentially expressed genes and quantified their expression by real-time polymerase chain reaction in peripheral blood of MCL patients (n=21), purified MCL cells (n=6) and naive B cells (n=4), obtaining fully concordant results. A computer-assisted approach was used to procure specific molecular signalling pathways that were aberrantly expressed in MCL cells. Several genes related to apoptosis and to the PI3K/AKT, WNT and tumour growth factor beta signalling pathways were altered in MCL cells when compared with naive B cells. These pathways may play a significant role in the pathogenesis of MCL and deserve further investigation as candidates for new therapeutic targets.

Heavy metals, depending on their oxidation states, can be highly reactive and, as a consequence, toxic to most organisms. They are produced by an expanding variety of anthropogenic sources suggesting an increasingly important role for this form of pollution. The toxic effect of heavy metals appears to be related to production of reactive oxygen species (ROS) and the resulting unbalanced cellular redox status. Algae respond to heavy metals by induction of several antioxidants, including diverse enzymes such as superoxide dismutase, catalase, glutathione peroxidase and ascorbate peroxidase, and the synthesis of low molecular weight compounds such as carotenoids and glutathione. At high, or acute, levels of metal pollutants, damage to algal cells occurs because ROS levels exceed the capacity of the cell to cope. At lower, or chronic, levels algae accumulate heavy metals and can pass them on to organisms of other trophic levels such as mollusks, crustaceans, and fishes. We review here the evidence linking metal accumulation, cellular toxicity, and the generation of ROS in aquatic environments.

The ability of phytoplankton to cope with oxidative stress is one of the main factors that influence its survival in the marine environment, when senescence conditions prevail. In a first attempt to investigate the antioxidant strategies of different phytoplanktonic groups face to oxidative stress, the superoxide dismutase (SOD; EC 1.15.1.1) activity and photosynthetic pigment content along the growth curves of the dinoflagellate Lingulodinium polyedrum (Stein) Dodge, the prasinophycean Tetraselmis gracilis (Kylin) Butcher and the diatom Minutocellus polymorphus (Hargraves and Guillard) Hasle, von Stosch and Syvertsen were evaluated in batch-cultures. Total SOD activity was determined by an indirect method involving the inhibition of cytochrome c reduction. The contents of photosynthetic pigments were analysed by HPLC using a reverse phase column (RP-18), based on a ternary gradient. A peak of total SOD activity was detected at the beginning of the T. gracilis and M. polymorphus exponential growth. In L. polyedrum and M. polymorphus, SOD activity increased approximately three times by day 17 of growth, compared to the values obtained on day 3 (exponential phase) of the growth curve. All three species of microalgae had reduced SOD activity at the end of their growth. The levels of peridinin in L. polyedrum increased about 60% by day 17 of growth compared to the values obtained at exponential phase. Tetraselmis gracilis exhibited a remarkable increase (approximately 85%) in beta-carotene concentration after 10-14 days of growth whereas the beta-carotene levels in M. polymorphus decreased about 85% along its growth curve. These findings suggest that the antioxidant response during senescence in batch-cultures differ according to the species. Induction of SOD activity may occur either in the early exponential or stationary growth phases, which is important to prevent oxidative stress triggered by a number of factors that affects growth, such as nutrient and light availability.

Superoxide dismutase (SOD) was studied in the agarophyte Gracilariopsis tenuifrons. Similar SOD activity (130 ± 9Um g −1 ) was observed in material from different regions of South America, from different phases of the life cycle (gametophytes and tetrasporophytes), and from apical and basal sections of the thallus. In alga grown under a light-dark cycle, SOD activity in samples taken at different times exhibited a diurnal rhythm. The activity measured during the day phase was twice as much as during the night phase. This rhythm did not persist under constant light, indicating light regulation of SOD activity. SOD activity was tested in algae submitted to different light intensities and different wavelengths. It increased with the light intensity. The blue light wavelength exerted a greater induction of SOD activity than other specific wavelengths.

Tumorigenicity of human pluripotent stem cells is a major threat limiting their application in cell therapy protocols. It remains unclear, however, whether suppression of tumorigenic potential can be achieved without critically affecting pluripotency. A previous study has identified hyperexpressed genes in cancer stem cells, among which is E2F2, a gene involved in malignant transformation and stem cell self-renewal. Here we tested whether E2F2 knockdown would affect the proliferative capacity and tumorigenicity of human embryonic stem cells (hESC). Transient E2F2 silencing in hESC significantly inhibited expression of the proto-oncogenes BMI1 and HMGA1, in addition to proliferation of hESC, indicated by a higher proportion of cells in G1, fewer cells in G2/M phase, and a reduced capacity to generate hESC colonies in vitro. Nonetheless, E2F2-silenced cells kept expression of typical pluripotency markers and displayed differentiation capacity in vitro. More importantly, E2F2 knockdown in hESC significantly inhibited tumor growth in vivo, which was considerably smaller than tumors generated from control hESC, although displaying typical teratoma traits, a major indicator of pluripotency retention in E2F2-silenced cells. These results suggest that E2F2 knockdown can inhibit hESC proliferation and tumorigenicity without significantly harming stemness, providing a rationale to future protocols aiming at minimizing risks related to therapeutic application of cells and/or products derived from human pluripotent cells.

A circadian rhythm in the total activity of superoxide dismutase (SOD; EC 1.15.1.1) from the unicellular alga Lingulodinium polyedrum is shown to be attributable to the mitochondrial MnSOD and chloroplastic FeSOD isoforms. Activity gels and labelling with polyclonal antibodies against pure CuZnSOD, MnSOD and FeSOD revealed a distinct circadian pattern in the abundance of the latter two isoforms, with peak values in early photophase 5 times greater than at the dark phase. However, no such changes were detected for the CuZnSOD isoform, which remained at constant levels over the 24-h light/dark cycle. These SOD isoforms might provide protection against damage from photochemically generated oxygen radicals, thus preventing subcellular oxidative stress.

Isolated chloroplasts from the unicellular dinoflagellate Gonyaulax polyedra were obtained from cells at LD:03 and LD:15 of cell cultures conditioned to light/dark cycles of 12h/12h and 19 o C temperature. Kinase activity in these organelles at different times of the day was estimated by phosphorylation experiments with radioactive labelled ATP. [?P 32]-ATP was incubated with intact organelles and the incorporation rate to proteins were estimated by autoradiography after a SDS-PAGE chromatography. Low molecular mass polypeptides were phosphorylated at night phase more intensively than in those day-is olated plastids.

The combination of cell therapy with growth factors could be a useful approach to treat progressive muscular dystrophies. Here, we demonstrate, for the first time, that IGF-1 considerably enhances the myogenesis of human umbilical cord (UC) mesenchymal stromal cells (MSCs) in vitro and that IGF-1 enhances interaction and restoration of dystrophin expression in co-cultures of MSCs and muscle cells from Duchenne patients. In vivo studies showed that human MSCs were able to reach the skeletal muscle of LAMA2(dy/2j) dystrophic mice, through systemic delivery, without immunosuppression. Moreover, we showed, for the first time, that IGF-1 injected systemically together with MSCs markedly reduced muscle inflammation and fibrosis, and significantly improved muscle strength in dystrophic mice. Our results suggest that a combined treatment with IGF-1 and MSCs enhances efficiency of muscle repair and, therefore, should be further considered as a potential therapeutic approach in muscular dystrophies.

NaPi2b, a sodium-dependent phosphate transporter, is highly expressed in ovarian carcinomas and is recognized by the murine monoclonal antibody MX35. The antibody had shown excellent targeting to ovarian cancer in several early phase clinical trials but being murine the antibody's full therapeutic potential could not be explored. To overcome this impediment we developed a humanized antibody version named Rebmab200, expressed in human PER.C6® cells and cloned by limiting dilution. In order to select a clone with high therapeutic potential clones were characterized using a series of physicochemical assays, flow cytometry, real-time surface plasmon resonance, glycosylation analyses, immunohistochemistry, antibody-dependent cell-mediated cytotoxicity, complement-dependent-cytotoxicity assays and quantitative PCR. Comparative analyses of Rebmab200 and MX35 monoclonal antibodies demonstrated that the two antibodies had similar specificity for NaPi2b by flow cytometry with a panel of 30 cell lines and maintained similar kinetic parameters. Robust and high producer cell clones potentially suitable for use in manufacturing were obtained. Rebmab200 antibodies were assessed by immunohistochemistry using a large panel of tissues including human carcinomas of ovarian, lung, kidney and breast origin. An assessment of its binding towards 33 normal human organs was performed as well. Rebmab200 showed selected strong reactivity with the tested tumor types but little or no reactivity with the normal tissues tested confirming its potential for targeted therapeutics strategies. The remarkable cytotoxicity shown by Rebmab200 in OVCAR-3 cells is a significant addition to the traits of stability and productivity displayed by the top clones of Rebmab200. Antibody-dependent cell-mediated toxicity functionality was confirmed in repeated assays using cancer cell lines derived from ovary, kidney and lung as targets. To explore use of this antibody in clinical trials, GMP production of Rebmab200 has been initiated. As the next step of development, Phase I clinical trials are now planned for translation of Rebmab200 into the clinic.

Marine planktonic algae are frequently exposed to metallic contaminants. Because heavy metals can be assimilated and accumulated by algal cells, they can then be transferred to higher trophic levels of food chains. We studied the effects of cadmium on protein production and the growth of the marine prasinophyte Tetraselmis gracilis (Kylin) Butcher. By means of toxicological assays, we estimated the LC{sub 50} of cadmium as 3.2 ppm and 1.8 ppm after 48 h and 96 h of exposure to this heavy metal, respectively. The growth of curves and survival percentages of cell cultures in the presence of cadmium were determined, and a proportional reduction of both parameters with increasing metal concentrations of cadmium, T. gracilis contained high levels of superoxide dismutase (SOD) activity, one of the main enzymes of the cell`s antioxidant defense mechanism. Under these growth conditions, total SOD activity in crude extracts was increased by 41% (at 1.5 ppm) and 107% (at 3.0 ppm). Assays of SOD activity in nondenaturing polyacrylamide gels also showed a similar induction by cadmium. These results show that cadmium has potentially toxic properties since it significantly inhibited the growth of T. gracilis at low concentrations and promoted by induction of SOD activity, suggestivemore » of an oxidative stress state. Besides being the first report of SOD in T. gracilis, this work describes experimental evidence of SOD induction by cadmium in this species. 56 refs., 4 figs., 1 tab.« less

Article history: Received on: 03/09/2012 Revised on: 14/09/2012 Accepted on: 20/09/2012 Available online: 28/09/2012 Plant-derived substances have traditionally played important roles in the treatment of human diseases, including of great significance to cancer therapy. Plants of the genus Pterodon (Fabaceae, Leguminosae), commonly known as ‘sucupira’, are disseminated throughout the central region of Brazil and have been used frequently in popular medicine. In recent years, interest in these plants has increased considerably. The biological effects of their extracts and pure metabolites have been investigated in several experimental models in vivo and in vitro. Until the present day, the antitumor effect of Pterodon plants on brain tumors is unknown. Therefore, the aim of this work was to investigate the action of P. emarginatus Vogel extracts and its fractions on glioblastoma cells. The hexane (HE), dichloromethane (DE) and ethanol (EE) extracts were obtained from seeds powder in each solvent. The diterpene 14,15-epoxygeranylgeraniol was obtained from HE fractionation. For tumorigenic assays, the extracts and fractions were added to U87MG, a human glioblastoma cells line. The cell viability assay showed that the proliferation of U87MG was inhibited by both extracts and the 14,15epoxygeranylgeraniol. Further trials in vivo will help to confirm these results, and may contribute to generate natural compounds for the treatment of this type of cancer.

All living cells utilize intricate DNA repair mechanisms to address numerous types of DNA lesions and to preserve genomic integrity, and pluripotent stem cells have specific needs due to their remarkable ability of self-renewal and differentiation into different functional cell types. Not surprisingly, human stem cells possess a highly efficient DNA repair network that becomes less efficient upon differentiation. Moreover, these cells also have an anaerobic metabolism, which reduces the mitochondria number and the likelihood of oxidative stress, which is highly related to genomic instability. If DNA lesions are not repaired, human stem cells easily undergo senescence, cell death or differentiation, as part of their DNA damage response, avoiding the propagation of stem cells carrying mutations and genomic alterations. Interestingly, cancer stem cells and typical stem cells share not only the differentiation potential but also their capacity to respond to DNA damage, with important implications for cancer therapy using genotoxic agents. On the other hand, the preservation of the adult stem cell pool, and the ability of cells to deal with DNA damage, is essential for normal development, reducing processes of neurodegeneration and premature aging, as one can observe on clinical phenotypes of many human genetic diseases with defects in DNA repair processes. Finally, several recent findings suggest that DNA repair also plays a fundamental role in maintaining the pluripotency and differentiation potential of embryonic stem cells, as well as that of induced pluripotent stem (iPS) cells. DNA repair processes also seem to be necessary for the reprogramming of human cells when iPS cells are produced. Thus, the understanding of how cultured pluripotent stem cells ensure the genetic stability are highly relevant for their safe therapeutic application, at the same time that cellular therapy is a hope for DNA repair deficient patients.

Introduction Ovarian adenocarcinoma is frequently detected at the late stage, when therapy efficacy is limited and death occurs in up to 50% of the cases. A potential novel treatment for this disease is a monoclonal antibody that recognizes phosphate transporter sodium-dependent phosphate transporter protein 2b (NaPi2b). Materials and methods To better understand the expression of this protein in different histologic types of ovarian carcinomas, we immunostained 50 tumor samples with anti-NaPi2b monoclonal antibody MX35 and, in parallel, we assessed the expression of the gene encoding NaPi2b (SCL34A2) by in silico analysis of microarray data. Results Both approaches detected higher expression of NaPi2b (SCL34A2) in ovarian carcinoma than in normal tissue. Moreover, a comprehensive analysis indicates that SCL34A2 is the only gene of the several phosphate transporters genes whose expression differentiates normal from carcinoma samples, suggesting it might exert a major role in ovarian carcinomas. Immunohistochemical and mRNA expression data have also shown that 2 histologic subtypes of ovarian carcinoma express particularly high levels of NaPi2b: serous and clear cell adenocarcinomas. Serous adenocarcinomas are the most frequent, contrasting with clear cell carcinomas, rare, and with worse prognosis. Conclusion This identification of subgroups of patients expressing NaPi2b may be important in selecting cohorts who most likely should be included in future clinical trials, as a recently generated humanized version of MX35 has been developed.

Autophagy is a lysosome-dependent degradation pathway that allows cells to recycle damaged or superfluous cytoplasmic content, such as proteins, organelles, and lipids. As a consequence of autophagy, the cells generate metabolic precursors for macromolecular biosynthesis or ATP generation. Deficiencies in this pathway were associated to several pathological conditions, such as neurodegenerative and cardiac diseases, cancer, and aging. The aim of this review is to summarize recent discoveries showing that autophagy also plays a critical role in stem cell maintenance and in a variety of cell differentiation processes. We also discuss a possible role for autophagy during cellular reprogramming and induced pluripotent stem (iPS) cell generation by taking advantage of ATP generation for chromatin remodeling enzyme activity and mitophagy. Finally, the significance of autophagy modulation is discussed in terms of augmenting efficiency of iPS cell generation and differentiation processes.

Progress in human genomics and development of high-throughput techniques to screen mutations, chromosomal aberrations, epigenetic alterations, and differential gene and microRNA expression profiles have brought a new dimension to stem cell and cancer research. Since the identification of biologically distinct populations of stem-like cells in a variety of human cancers, in which they are associated with the initial steps of tumorigenesis, a new wave of studies have been focusing on the detailed molecular and genetic characterization of such cancer stem cells (CSCs). Molecular studies on CSCs and respective normal counterparts shall help the identification of precancerous alterations, putative new oncogenes, and tumor suppressors. Elucidation of the cellular and molecular nature of tumors and information about the initial steps toward malignant transformation are examples of scientific contribution that are expected to emerge from this field of intense research. Based on recent advances in stem cell research, a brief description of the main genes and pathways relevant to the biology of CSCs is discussed in this chapter. More specifically, the chapter focuses on genetic and epigenetic alterations affecting expression of genes regulating typical stem cell processes such as self-renewal, pluripotency, and differentiation, and their correlations with the tumor-initiating cell phenotype. Detailed and updated information on stem cell molecular biology and genomics can be found online in public databases such as the Stem Cell Database of Mount Sinai School of Medicine and the University of Pennsylvania (http://stemcell.mssm.edu/v2/) and the NIH Stem Cell Unit (http://explore.ninds.nih.gov/scudbp/public.do).

Aberrant expression of stem cell-related genes in tumors may confer more primitive and aggressive traits affecting clinical outcome. Here, we investigated expression and prognostic value of the neural stem cell marker CD133, as well as of the pluripotency genes LIN28 and OCT4 in 37 samples of pediatric medulloblastoma, the most common and challenging type of embryonal tumor. While most medulloblastoma samples expressed CD133 and LIN28, OCT4 expression was found to be more sporadic, with detectable levels occurring in 48% of tumors. Expression levels of OCT4, but not CD133 or LIN28, were significantly correlated with shorter survival (P ≤ 0.0001). Median survival time of patients with tumors hyperexpressing OCT4 and tumors displaying low/undetectable OCT4 expression were 6 and 153 months, respectively. More importantly, when patients were clinically stratified according to their risk of tumor recurrence, positive OCT4 expression in primary tumor specimens could discriminate patients classified as average risk but which further deceased within 5 years of diagnosis (median survival time of 28 months), a poor clinical outcome typical of high risk patients. Our findings reveal a previously unknown prognostic value for OCT4 expression status in medulloblastoma, which might be used as a further indicator of poor survival and aid postoperative treatment selection, with a particular potential benefit for clinically average risk patients.

In the developing cerebellum, proliferation of granular neuroprogenitor (GNP) cells lasts until the early postnatal stages when terminal maturation of the cerebellar cortex occurs. GNPs are considered cell targets for neoplastic transformation, and disturbances in cerebellar GNP cell proliferation may contribute to the development of pediatric medulloblastoma. At the molecular level, proliferation of GNPs is regulated through an orchestrated action of the SHH, NOTCH, and WNT pathways, but the underlying mechanisms still need to be dissected. Here, we report that expression of the E2F1 transcription factor in rat GNPs is inversely correlated with cell proliferation rate during postnatal development, as opposed to its traditional SHH-dependent induction of cell cycle. Proliferation of GNPs peaked at postnatal day 3 (P3), with a subsequent continuing decrease in proliferation rates occurring until P12. Such gradual decline in proliferating neuroprogenitors paralleled the extent of cerebellum maturation confirmed by histological analysis with cresyl violet staining and temporal expression profiling of SHH, NOTCH2, and WNT4 genes. A time course analysis of E2F1 expression in GNPs revealed significantly increased levels at P12, correlating with decreased cell proliferation. Expression of the cell cycle inhibitor p18 ( Ink4c ), a target of E2F1, was also significantly higher at P12. Conversely, increased E2F1 expression did not correlate with either SMAC/DIABLO and BCL2 expression profiles or apoptosis of cerebellar cells. Altogether, these results suggest that E2F1 may also be involved in the inhibition of GNP proliferation during rat postnatal development despite its conventional mitogenic effects.

Meduloblastoma é um tumor maligno do sistema nervoso central (SNC). Na infância, representa o tumor sólido mais frequente e a principal causa de morte relacionada ao câncer. Tratamentos atuais incluem cirurgia, quimioterapia e radioterapia, que podem trazer prejuízos cognitivos e desenvolvimento de tumores secundários. Novas perspectivas terapêuticas surgem com a identificação de células-tronco em gliomas, as quais apresentam alto potencial tumorigênico e maior resistência à radioterapia e quimioterapia. A hipótese das células-tronco tumorais sugere que a transformação de células-tronco e/ou progenitores neurais do cerebelo está envolvida no desenvolvimento do meduloblastoma. Portanto, analisar alterações genéticas e moleculares envolvidas nesse processo é de grande importância na pesquisa básica e aplicada ao câncer. Nesse sentido, discutimos o possível envolvimento de vias de sinalização bioquímica críticas a ambos os processos de neurogênese normal ou tumorigênese, com base em evidências atuais na área de genética e biologia molecular dos meduloblastomas. Do ponto de vista clínico, a modulação de vias de sinalização como a do TGFβ, regulando proliferação de célula-tronco neural e desenvolvimento tumoral, pode ser uma estratégia alternativa para o desenvolvimento de novos medicamentos objetivando-se terapias mais eficientes e melhora do prognóstico dos pacientes pediátricos com câncer de SNC. Medulloblastoma is a highly malignant primary tumor of the central nervous system. It represents the most frequent type of solid tumor and the leading cause of death related to cancer in early childhood. Current treatment includes surgery, chemotherapy and radiotherapy which may lead to severe cognitive impairment and secondary brain tumors. New perspectives for therapeutic development have emerged with the identification of stem-like cells displaying high tumorigenic potential and increased radio- and chemo-resistance in gliomas. Under the cancer stem cell hypothesis, transformation of neural stem cells and/or granular neuron progenitors of the cerebellum are though to be involved in medulloblastoma development. Dissecting the genetic and molecular alterations associated with this process should significantly impact both basic and applied cancer research. Based on cumulative evidences in the fields of genetics and molecular biology of medulloblastomas, we discuss the possible involvement of developmental signaling pathways as critical biochemical switches determining normal neurogenesis or tumorigenesis. From the clinical viewpoint, modulation of signaling pathways such as TGFβ, regulating neural stem cell proliferation and tumor development, might be attempted as an alternative strategy for future drug development aiming at more efficient therapies and improved clinical outcome of patients with pediatric brain cancers. Universidade Federal de São Paulo (UNIFESP) Laboratory of Experimental Neurology Department of Neurology and Neurosurgery UNIFESP Laboratory of Experimental Neurology Department of Neurology and Neurosurgery UNIFESP GRAACC Instituto de Oncologia Pediátrica UNIFESP, Laboratory of Experimental Neurology Department of Neurology and Neurosurgery UNIFESP, Laboratory of Experimental Neurology Department of Neurology and Neurosurgery UNIFESP, GRAACC Instituto de Oncologia Pediátrica SciELO

Background A higher burden of head and neck cancer has been reported to affect deprived populations. This study assessed the association between socioeconomic status and head and neck cancer, aiming to explore how this association is related to differences of tobacco and alcohol consumption across socioeconomic strata. Methods We conducted a case-control study in Sao Paulo, Brazil (1998–2006), including 1017 incident cases of oral, pharyngeal and laryngeal cancer, and 951 sex- and age-matched controls. Education and occupation were distal determinants in the hierarchical approach; cumulative exposure to tobacco and alcohol were proximal risk factors. Outcomes of the hierarchical model were compared with fully adjusted ORs. Results Individuals with lower education (OR 2.27; 95% CI 1.61 to 3.19) and those performing manual labour (OR 1.55; 95% CI 1.26 to 1.92) had a higher risk of disease. However, 54% of the association with lower education and 45% of the association with manual labour were explained by proximal lifestyle exposures, and socioeconomic status remained significantly associated with disease when adjusted for smoking and alcohol consumption. Conclusions Socioeconomic differences in head and neck cancer are partially attributable to the distribution of tobacco smoking and alcohol consumption across socioeconomic strata. Additional mediating factors may explain the remaining variation of socioeconomic status on head and neck cancer.

Osteosarcoma (OS) is the most frequent bone tumor in children and adolescents. Tumor antigens are encoded by genes that are expressed in many types of solid tumors but are silent in normal tissues, with the exception of placenta and male germ-line cells. It has been proposed that antigen tumors are potential tumor markers.The premise of this study is that the identification of novel OS-associated transcripts will lead to a better understanding of the events involved in OS pathogenesis and biology.We analyzed the expression of a panel of seven tumor antigens in OS samples to identify possible tumor markers. After selecting the tumor antigen expressed in most samples of the panel, gene expression profiling was used to identify osteosarcoma-associated molecular alterations. A microarray was employed because of its ability to accurately produce comprehensive expression profiles.PRAME was identified as the tumor antigen expressed in most OS samples; it was detected in 68% of the cases. Microarray results showed differences in expression for genes functioning in cell signaling and adhesion as well as extracellular matrix-related genes, implying that such tumors could indeed differ in regard to distinct patterns of tumorigenesis.The hypothesis inferred in this study was gathered mostly from available data concerning other kinds of tumors. There is circumstantial evidence that PRAME expression might be related to distinct patterns of tumorigenesis. Further investigation is needed to validate the differential expression of genes belonging to tumorigenesis-related pathways in PRAME-positive and PRAME-negative tumors.

Adult or somatic stem cells hold great promise for tissue regeneration. Currently, one major scientific interest is focused on the basic biology and clinical application of mesenchymal stem cells. Adipose tissue-derived stem cells share similar characteristics with bone marrow mesenchymal stem cells, but have some advantages including harvesting through a less invasive surgical procedure. Moreover, adipose tissue-derived stem cells have the potential to differentiate into cells of mesodermal origin, such as adipocytes, cartilage, bone, and skeletal muscle, as well as cells of non-mesodermal lineage, such as hepatocytes, pancreatic endocrine cells, neurons, cardiomyocytes, and vascular endothelial cells. There are, however, inconsistencies in the scientific literature regarding methods for harvesting adipose tissue and for isolating, characterizing and handling adipose tissue-derived stem cells. Future clinical applications of adipose tissue-derived stem cells rely on more defined and widespread methods for obtaining cells of clinical grade quality. In this review, current methods in adipose tissue-derived stem cell research are discussed with emphasis on strategies designed for future applications in regenerative medicine and possible challenges along the way.

Background Uncovering the molecular mechanisms involved in epileptogenesis is critical to better understand the physiopathology of epilepsies and to help develop new therapeutic strategies for this prevalent and severe neurological condition that affects millions of people worldwide. Results Changes in the transcriptome of hippocampal cells from rats subjected to the pilocarpine model of epilepsy were evaluated by microarrays covering 34,000 transcripts representing all annotated rat genes to date. Using such genome-wide approach, differential expression of nearly 1,400 genes was detected during the course of epileptogenesis, from the early events post status epilepticus (SE) to the onset of recurrent spontaneous seizures. Most of these genes are novel and displayed an up-regulation after SE. Noteworthy, a group of 128 genes was found consistently hyper-expressed throughout epileptogenesis, indicating stable modulation of the p38MAPK, Jak-STAT, PI3K, and mTOR signaling pathways. In particular, up-regulation of genes from the TGF-beta and IGF-1 signaling pathways, with opposite effects on neurogenesis, correlate with the physiopathological changes reported in humans. Conclusions A consistent regulation of genes functioning in intracellular signal transduction regulating neurogenesis have been identified during epileptogenesis, some of which with parallel expression patterns reported in patients with epilepsy, strengthening the link between these processes and development of epilepsy. These findings reveal dynamic molecular changes occurring in the hippocampus that may serve as a starting point for designing alternative therapeutic strategies to prevent the development of epilepsy after acquired brain insults.

Bone deposition and bone resorption are ongoing dynamic processes, constituting bone remodeling. Some bone tumors, such as osteosarcoma (OS), stimulate focal bone deposition. OS is the most common primary bone tumor in children and young adults. A complex network of genes regulates bone remodeling and alterations in its expression levels can influence the genesis and progression of bone diseases, including OS. We hypothesized that the expression profiles of bone remodeling regulator genes would be correlated with OS biology and clinical features. We used real-time PCR to evaluate the mRNA levels of the tartrate-resistant acid phosphatase (ACP5), colony stimulating factor-1 (CSF1R), bone morphogenetic protein 7 (BMP7), collagen, type XI, alpha 2 (COL11A2), and protein tyrosine phosphatases zeta 1 (PTPRZ1) genes, in 30 OS tumor samples and correlated with clinical and histological data. All genes analyzed, except CSF1R, were differentially expressed when compared with normal bone expression profiles. In our results, OS patients with high levels of COL11A2 mRNA showed worse overall (p = 0.041) and event free survival (p = 0.037). Also, a trend for better overall survival was observed in patients with samples showing higher expression of BMP7 (p = 0.067). COL11A2 overexpression and BMP7 underexpression could collaborate to OS tumor growth, through its central role in bone remodeling process.

Mesenchymal stem cells (MSC) are multipotent cells which can be obtained from several adult and fetal tissues including human umbilical cord units. We have recently shown that umbilical cord tissue (UC) is richer in MSC than umbilical cord blood (UCB) but their origin and characteristics in blood as compared to the cord remains unknown. Here we compared, for the first time, the exonic protein-coding and intronic noncoding RNA (ncRNA) expression profiles of MSC from match-paired UC and UCB samples, harvested from the same donors, processed simultaneously and under the same culture conditions. The patterns of intronic ncRNA expression in MSC from UC and UCB paired units were highly similar, indicative of their common donor origin. The respective exonic protein-coding transcript expression profiles, however, were significantly different. Hierarchical clustering based on protein-coding expression similarities grouped MSC according to their tissue location rather than original donor. Genes related to systems development, osteogenesis and immune system were expressed at higher levels in UCB, whereas genes related to cell adhesion, morphogenesis, secretion, angiogenesis and neurogenesis were more expressed in UC cells. These molecular differences verified in tissue-specific MSC gene expression may reflect functional activities influenced by distinct niches and should be considered when developing clinical protocols involving MSC from different sources. In addition, these findings reinforce our previous suggestion on the importance of banking the whole umbilical cord unit for research or future therapeutic use. Electronic supplementary material The online version of this article (doi:10.1007/s12015-009-9098-5) contains supplementary material, which is available to authorized users.

Aims: Annexin A1 (ANXA1) is a soluble cytoplasmic protein, moving to membranes when calcium levels are elevated. ANXA1 has also been shown to move to the nucleus or outside the cells, depending on tyrosine-kinase signalling, thus interfering in cytoskeletal organization and cell differentiation, mostly in inflammatory and neoplastic processes. The aim was to investigate subcellular patterns of immunohistochemical expression of ANXA1 in neoplastic and non-neoplastic samples from patients with laryngeal squamous cell carcinomas (LSCC), to elucidate the role of ANXA1 in laryngeal carcinogenesis. Methods and results: Serial analysis of gene expression experiments detected reduced expression of ANXA1 gene in LSCC compared with the corresponding non-neoplastic margins. Quantitative polymerase chain reaction confirmed ANXA1 low expression in 15 LSCC and eight matched normal samples. Thus, we investigated subcellular patterns of immunohistochemical expression of ANXA1 in 241 paraffin-embedded samples from 95 patients with LSCC. The results showed ANXA1 down-regulation in dysplastic, tumourous and metastatic lesions and provided evidence for the progressive migration of ANXA1 from the nucleus towards the membrane during laryngeal tumorigenesis. Conclusions: ANXA1 dysregulation was observed early in laryngeal carcinogenesis, in intra-epithelial neoplasms; it was not found related to prognostic parameters, such as nodal metastases.

Sociedade Beneficente Israelita Brasileira Hospital Albert Einstein SBIBHAE Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) CD133 antigen is an integral membrane glycoprotein that can bind with different cells. Originally, however. this cellular surface antigen was expressed in human stem cells and in various cellular progenitors of the haematopoietic system. Human cord blood has been described as an excellent source of CD133(+) haematopoietic progenitor cells with a large application potential. One of the main objectives of the present study is to describe for the first time the ultrastructural characteristics of CD133(+) stem cells using transmission electronic microscopy. Another objective of the manuscript is to demonstrate through transmission electronic microscopy the molecular image of magnetic nanoparticles connected to the stein cells of great biotechnological importance, as well as demonstrating the value of this finding for electronic paramagnetic resonance and its related nanobioscientific value. Ultrastructural results showed the monoclonal antibody anti-CD133 bound to the superparamagnetic nanoparticles by the presence of electrondense granules in cell membrane, as well as in the cytoplasm, revealing the ultrastructural characteristics of CD133(+) cells, exhibiting a round morphology with discrete cytoplasmic projections, having an active nucleus that follows this morphology. the cellular cytoplasm was filled up with mitochondrias, as well as microtubules and vesicles pinocitic. characterizing the process as being related to internalization of the magnetic nanoparticles that were endocyted by the cells in question. Electronic paramagnetic resonance analysis of the CD133(+) stem cells detected that the small (spectrum) generated by the labelled cells comes from the superparamagnetic nanoparticles that are bound to them. These results strongly suggest that these CD133(+) cells can be used in nanobiotechnology applications, with benefits in different biomedical areas. Albert Einstein Res & Educ Inst IEPAE, São Paulo, Brazil Univ São Paulo, Biotechnol Program, IPT, Butanan Inst, São Paulo, Brazil Univ São Paulo, Dept Radiol, São Paulo, Brazil Univ São Paulo, Dept Immunol, Inst Biomed Sci, São Paulo, Brazil UNESP, Biosci Inst, Dept Biol, Rio Claro, SP, Brazil Universidade Federal de São Paulo, Dept Neurol & Neurosurg, São Paulo, Brazil Universidade Federal de São Paulo, Dept Neurol & Neurosurg, São Paulo, Brazil Sociedade Beneficente Israelita Brasileira Hospital Albert Einstein SBIBHAE: (IIEP-AE 278-07 FAPESP: 02/01826-5 Web of Science

We have performed cDNA microarray analyses to identify gene expression differences between highly invasive glioblastoma multiforme (GBM) and typically benign pilocytic astrocytomas (PA). Despite the significant clinical and pathological differences between the 2 tumor types, only 63 genes were found to exhibit 2-fold or greater overexpression in GBM as compared to PA. Forty percent of these genes are related to the regulation of the cell cycle and mitosis. QT-PCR validation of 6 overexpressed genes: MELK, AUKB, ASPM, PRC1, IL13RA2 and KIAA0101 confirmed at least a 5-fold increase in the average expression levels in GBM. Maternal embryonic leucine zipper kinase (MELK) exhibited the most statistically significant difference. A more detailed investigation of MELK expression was undertaken to study its oncogenic relevance. In the examination of more than 100 tumors of the central nervous system, we found progressively higher expression of MELK with astrocytoma grade and a noteworthy uniformity of high level expression in GBM. Similar level of overexpression was also observed in medulloblastoma. We found neither gene promoter hypomethylation nor amplification to be a factor in MELK expression, but were able to demonstrate that MELK knockdown in malignant astrocytoma cell lines caused a reduction in proliferation and anchorage-independent growth in in vitro assays. Our results indicate that GBM and PA differ by the expression of surprisingly few genes. Among them, MELK correlated with malignancy grade in astrocytomas and represents a therapeutic target for the management of the most frequent brain tumors in adult and children.

The identification of mesenchymal stem cell (MSC) sources that are easily obtainable is of utmost importance. Several studies have shown that MSCs could be isolated from umbilical cord (UC) units. However, the presence of MSCs in umbilical cord blood (UCB) is controversial. A possible explanation for the low efficiency of MSCs from UCB is the use of different culture conditions by independent studies. Here, we compared the efficiency in obtaining MSCs from unrelated paired UCB and UC samples harvested from the same donors. Samples were processed simultaneously, under the same culture conditions. Although MSCs from blood were obtained from only 1 of the 10 samples, we were able to isolate large amounts of multipotent MSCs from all UC samples, which were able to originate different cell lineages. Since the routine procedure in UC banks has been to store the blood and discard other tissues, such as the cord and/or placenta, we believe our results are of immediate clinical value. Furthermore, the possibility of originating different cell lines from the UC of neonates born with genetic defects may provide new cellular research models for understanding human malformations and genetic disorders, as well as the possibility of testing the effects of different therapeutic drugs. Disclosure of potential conflicts of interest is found at the end of this article.

Pleiotrophin (PTN) is a secreted cytokine with several properties related with tumor development, including differentiation, angiogenesis, invasion, apoptosis and metastasis. There is evidence that PTN has also a relevant role in primary brain neoplasms and its inactivation could be important to treatment response. Astrocytic and oligodendroglial tumors are the most frequent primary brain neoplasms. Astrocytic tumors are classified as pilocytic astrocytoma (PA), diffuse astrocytoma (DA), anaplastic astrocytoma (AA) and glioblastoma (GBM). Oligodendroglial tumors are classified as oligodendroglioma (O) and anaplastic oligodendroglioma (AO). The aim of the present study was to compare PTN expression, in astrocytomas and oligodendrogliomas and its association with the histological diagnosis, microvascular density, proliferate potential and clinical outcome.Seventy-eight central nervous system tumors were analyzed. The histological diagnosis in accordance with WHO classification was: 13PA, 18DA, 8AA, 15GBM, 16O and 8AO. Immunohistochemistry was realized with these specific antibodies: pleiotrophin, CD31 to microvascular density and Ki-67 to cell proliferation.PTN expression was significantly higher in GBM and AA when compared to PA and higher in GBM compared to DA. PTN expression did not differ between O and AO. Proliferate index and microvascular density were evaluated only in high grade tumors (AA, GBM and AO) divided in three groups according to PTN expression (low, intermediate and high). These results showed no statistical difference between PTN expression and index of cellular proliferation and neither to PTN expression and microvascular density. Overall survival (OS) analysis (months) showed similar results in high grade gliomas with different levels of PTN expression.Our results suggest that PTN expression is associated with histopathological grade of astrocytomas. Proliferation rate, microvascular density and overall survival do not seem to be associated with PTN expression.

Toxicity bioassays based on survival were carried out with cells of the marine dinoflagellate Gonyaulax polyedra exposed to mercury (Hg2+ ), cadmium (Cd2+), lead (Pb2+) and copper (Cu2+). The toxicity scale of these metals found was Hg2+ > Cu2+ > Cd2+ > Pb2+. Cells exposed to metals promptly underwent encystment, which is an important strategy for surviving metal exposure. Following 48 h exposure to Cu2+, complete excystment occurred within 96 h after reinoculation of cells in fresh metal-free media, and with Pb2+ partial recovery occurred in that time. Bioluminescence was affected by the metals in a dose-dependent manner primarily by increasing the frequency of flashing, but the glow emission was also altered with acute Cu2+ and Pb2+ treatments. Several physiological processes in G. polyedra are under circadian control. Chronic exposures to metals caused no substantial alterations in the circadian rhythm of bioluminescence glow, indicating that the biological clock of this dinoflagellate is not sensitive to these metals at the concentrations tested.

On 10th February 2007, English entrepreneur Sir Richard Branson, best known for his Virgin brand of over 360 companies, announced the establishment of a cord blood bank, the Virgin Health Bank, in London. What makes this bank different is a dual public–private approach. A fifth of the cord blood would be stored for private use for the child or a family member and the rest would be donated to the public part of the bank which will be accessible to anyone in the world who needs it, at no cost. Additionally, Branson has pledged to donate his 50% of proceeds from Virgin Health Bank to researchers investigating the potential of cord blood stem cells [1]. The idea for the dual bank emerged when he was visited by a senior director of the National Blood Center, asking for his support in a charitable role, because children were dying through lack of umbilical cord blood. Initially Sir Richard offered 3 million pounds to the National Health Service to help them increase their storage capacity for umbilical cord blood, but the center was not comfortable with accepting funds from private sources. So Sir Richard decided to set up a company to do the job. This is an initiative that should be applauded but we want to draw attention to a very important and urgent aspect. The routine procedure in umbilical cord banks has been to store the blood and discard other tissues, such as the cord and/or placenta, which is a much better source of mesenchymal stem cells than blood [2]. Umbilical cord

The response of superoxide dismutase (SOD) activity in the marine dinoflagellate Gonyaulax polyedra to chronic (5.0 ppb Hg, 0.5 ppm Cd, 2.0 ppm Pb and 0.1 ppm Cu, during 30 days) and acute (10.0 ppb Hg, 1.0 ppm Cd, 5.0 ppm Pb and 0.25 ppm Cu, during 48 hours) exposure to metals was investigated. Under chronic exposure to Hg, Cd, Pb, and Cu, total SOD activity of metal-treated cells increased during the first day of exposure to plateau levels of 134, 148, 127, and 139% of control values respectively. Under acute metal exposure, SOD activity increases were of similar magnitude but much more rapid (within several hours) and of shorter duration. In addition, assays for oxidative damage to lipids revealed high levels of lipid peroxidation in cells kept in either chronic or acute exposure to metals reaching values 2-fold greater than the control group. Changes in SOD activity were dependent on the metal, its concentration, and the time of exposure. Non-denaturing polyacrylamide gels revealed induction of Fe-SOD and Mn-SOD but not Cu-Zn-SOD isoforms in cells kept under acute exposure to metals. These results suggest that oxidative stress may be an important mediator of metal toxicity in algal systems, with SOD providing antioxidant protection.

Daily oscillations of both beta-carotene and superoxide dismutase (SOD) activity are related to the intracellular control of reactive oxygen species (ROS). It is well established that ROS are present in all aerobic cells. We studied the marine dinoflagellate Gonyaulax polyedra which has been extensively used as a model to understand the biological clock at the molecular level. beta-Carotene, besides suppressing singlet molecular oxygen (1O2), may act as a photoreceptor pigment in many photosynthetic cells. The levels of beta-carotene during the day phase were shown to be twice as high as during the night phase. The dose-response curve for light-induced carotenoid synthesis was linear for up to 45 min of light exposure, after which night phase cells contained the same levels of beta-carotene as day phase cells. Cells exposed to light pulses at different times during the dark period displayed the highest beta-carotene induction in the middle of the night. SOD activity of cell-free extracts of G. polyedra was three to four times higher during the day. This rhythm continued in cells kept in constant light, indicating that the regulation can be attributed to the cellular circadian clock. No-denaturing polyacrylamide gels revealed the presence of several SOD isoenzymes in G. polyedra, including CuZnSOD and MnSOD. Furthermore, G. polyedra SOD cross-reacts with a polyclonal antibody raised against SOD. In addition to being gene regulated by ROS concentration, G. polyedra SOD expression seems also to be under the control of the biological clock.